Focal gene expression profiling of stained FFPE tissues with spatial correlation to morphology

ABSTRACT

Expression profiling of FFPE sample areas smaller than 2 mm2 with single-cell sensitivity, correlated with tissue microenvironment morphology and neoplastic grade. An automated digital molecular pathology instrument for integrated imaging, immunohistochemical assessment, and processing samples for sequence detection assays. Software for instrument and sample control and analysis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of Ser. No. 15/387,650entitled Ligation Assays in Liquid Phase, filed Dec. 22, 2016 andpublished as US 2017/0101671, which is a continuation-in-part ofinternational application PCT/US16/14999, filed Jan. 26, 2016 andpublished as WO 2016/123154, which is a continuation-in-part of Ser. No.14/788,670, filed Jun. 30, 2015 and issued as U.S. Pat. No. 9,856,521 onJan. 2, 2018, which claims the benefit of priority of U.S. provisionalapplication Ser. No. 62/108,161, filed Jan. 27, 2015.

Parent application Ser. No. 15/387,650, is also a continuation-in-partof Ser. No. 14/788,670, filed Jun. 30, 2015 and issued as U.S. Pat. No.9,856,521 on Jan. 2, 2018, which claims the benefit of priority of U.S.provisional application Ser. No. 62/108,161, filed Jan. 27, 2015.

This application is also a continuation-in-part of internationalapplication PCT/US18/24206, entitled Modulation of Targets in CellularPathways Identified by Resolution of Stochastic Gene Expression, filedMar. 23, 2018, which claims the benefit of priority of U.S. provisionalapplication Ser. No. 62/475,796, filed Mar. 23, 2017.

The contents of the aforementioned applications are incorporated hereinin their entirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under grants R43 & R44ES024107, R43 & R44 HG007339, R43 & R44 HG008917, R43 & R44 HG007815,R33CA183699, awarded by the National Institutes of Health (NIH). Thegovernment has certain rights in the invention.

TECHNICAL FIELD

This invention relates to histology, and more particularly to assays fordetecting nucleic acid sequences in tissue samples.

SUMMARY OF THE INVENTION

The present invention provides methods, kits, instruments, and softwarefor profiling nucleic acid sequences in minute focal areas ofhistological samples, such as formalin-fixed, paraffin-embedded tissuespecimens (FFPEs).

The detection assays disclosed herein (in different versions, butcollectively TempO-Seq™ assays) enable gene expression to be profiledfrom areas 1 mm² and smaller focal areas of, for example, of 5 μm thickFFPE sections of normal and cancerous tissue to identify diseasebiomarkers and mechanistic pathways. The invention can also be performedin situ on slides by an automated slide stainer, followed by antibodystaining and/or H&E (hematoxylin and eosin) staining. Then, using adigital imaging platform such as the automated CellSensus™ digitalimaging platform of the invention, areas as small as 130 μm down to 30μm in diameter within the FFPE section can be profiled, permitting thegene expression data to be correlated directly to the specificmorphology of that focal area. Smaller and irregular areas of FFPE canalso be profiled. Any preparation on slides can be profiled, such ascells fixed to a surface, and the number of cells or amount of tissuecan be as little as a single cell or portion of a cell, such as aportion of a neuron.

Pathologists can use the instrument and software of the invention toselect areas to be profiled for marker expression during the course oftheir histologic examination of the section. Detection assay products(such as ligated detector oligonucleotides) can be recoveredautomatically by the instrument from the selected regions of interest.After transferring the products into PCR tubes, any remaining steps inthe detection assay can be completed, such as PCR amplification orpreparation for sequencing. Analysis of the sequencing data can becarried out automatically by the software to report results. In thepresent invention, laser capture and destruction of the tissue becomeunnecessary. The slides processed by the invention can be dried, treatedto stabilize or preserve the sample, or otherwise archived, andadditional areas can be sampled at a later date.

Replicate areas of matched normal versus cancerous tissue can besampled, measuring gene biomarkers of clinical utility. Gene expressionprofiles are presented for scraped areas of normal, high grade PIN(prostatic intraepithelial neoplasia), and cancer epithelium fromprostate cancer patients to perform the TempO-Seq™ assay on H&E-stainedFFPE samples. The single-cell sensitivity of the in situ protocol isdemonstrated by comparing profiles of single MCF-7 cells from aprocessed Cytospin slide to single cells collected by flow cytometry.The reproducibility of the assay is demonstrated for H&E-stained FFPEsamples, as well as the specificity of biomarker expression obtainedfrom profiling areas of stroma, normal and cancer epithelium. These datademonstrate that the automated CellSensus™ platform and assays enablecomplex molecular tests to be carried out by pathologists in their ownlabs, and render moot the issues of “% cancer” and the amount of patienttissue required for testing. They demonstrate that spatial resolutionand specificity result in greater biomarker specificity. The presentinvention brings extraction-free complex molecular testing of FFPEs intothe pathology lab and provides simplicity, focal spatial precision andcorrelation to morphology to the field of molecular pathology. While theresults presented use fixed tissue or cells on a slide, anysurface-adherent sample can be tested as long as it survives the washsteps and the intracellular nucleic acid to be measured is accessible tothe reagents.

H&E- or antibody-stained FFPEs can be assayed, providingwhole-transcriptome or focused panels of data using as little as 1 mm²area of a 5 mm section. Molecular profiling of high grade PIN adjacentto cancer versus cancer is consistent with adjacent high grade PIN beingcancer in situ. Slides can be processed though the in situ assay usingan automated stainer, and antibody or H&E staining can be performed onthe processed slides. Immunohistochemistry (IHC) assessment can becarried out and areas for automated profiling selected using theCellSensus™ digital molecular pathology platform. The sample can be anysurface-adherent sample, such as FFPE or cells. The in situ assay hassingle-cell sensitivity, even for measuring low-expressed genes. Thearea profiled is marked so that profiling data can be positivelycorrelated to the tissue microenvironment morphology. Accordingly, thespatial resolution results in biomarker specificity.

Accordingly, the present invention provides a method for detecting anucleic acid sequence from a selected area of a sample in situ,comprising in any order: imaging the sample for the presence or absenceof an analyte; selecting an area of the sample less than 2 mm² based onthe imaging; detecting a target nucleic acid sequence having adownstream region (DR) and an upstream region (UR). The detection stepis performed by contacting at least the selected area of the sample witha downstream detector oligo (DDO) comprising a DR′ portion that iscomplementary to the DR, and an upstream detector oligo (UDO) comprisinga UR′ portion that is complementary to the UR, ligating the DR′ and UR′if both are specifically hybridized to the DR and UR of a targetsequence, and collecting the ligation products from the selected area.As a result, the ligation product indicates the presence of the targetsequence in the selected area.

The invention also provides a method for detecting a neoplastic state ofa cell by performing the method of the invention where a first cancermarker sequence is detected in the cell. The invention also provides amethod for generating a gene expression profile for the selected area,for a plurality of target sequences. A disease state can be diagnosed byperforming the method, wherein the target sequence is detected in thearea of a morphological feature. The invention also provides kits ofdetector oligos and stains. The invention further provides an instrumenthaving an imaging component, a component for collecting ligationproducts from the selected area, and a component for transferring theproducts to an external container.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a representative ligation assay for detecting targetnucleic acid sequences. Briefly, downstream detector (DD) and upstreamdetector (UD) probe oligonucleotides are allowed to (a) hybridize to atarget sequence, having DR and UR regions, in a sample. For convenienceof identification, upstream regions are often underlined herein. Whilehybridized to the DR and UR of the target sequence, the DD is (b2)ligated selectively to the UR. Optionally, the DD is (b0) extended priorto (b2) ligation. The ligation product is optionally (c) amplified viaamplification regions P1 and P2′ by one or more primers, such as P1 andP2.

FIG. 2a shows an “anchored” version of the assay where the UD isconfigured with a second complementary region (UR2′ or “anchor”)separated by a noncomplementary region (CP1). The DD and UD canhybridize to a target sequence as in FIG. 2b , forming a hybridizationcomplex (HC) providing a substrate for ligation at the junction (L)between DR′ and UR′. In some methods, an optional nuclease, such as a3′- or 5′-single-stranded exonuclease, is provided at various stages toremove undesired or leftover reactants. After ligation, FIG. 2c showsthe ligation product (LP) can be amplified by primers to yieldamplification products (AP) in FIG. 2 d.

FIG. 3 shows a modified version of the TempO-Seq™ assay that can beperformed after antibody-staining, before flow cytometry sorting (FACS)and PCR.

FIG. 4 depicts steps for processing FFPE samples in the “standard”TempO-Seq™ FFPE protocol.

FIG. 5 compares expression between normal and PIN (prostaticintraepithelial neoplasia) versus normal and cancer, plotting forstatistically significant genes, as discussed in Example 5.

FIG. 6 illustrates an automated in situ assay process.

In FIG. 7, panel (A) shows the correlation of an assay of bulk 200 cellsversus a single FACS-sorted cell. Panel (B) shows the correlation of thesame 200-cell bulk and a single cell profiled using the CellSensus™instrument. Panel (C) shows correlation of one single cell isolated byFACS versus a single cell isolated by the CellSensus™ instrument.

FIG. 8 shows images of a breast FFPE before and after automated elutionby the CellSensus™ instrument, showing that a reagent in the eluentdestains the exposed area, providing a positive record of the profiledarea.

FIG. 9 shows stained prostate FFPE tissue (left) and the same tissueafter focal elution of a 130 μm diameter area by the CellSensus™instrument (right). The destained area in the center demonstratedexquisite elution and collection from minute spatial areas. Theprecision of the collection areas is demonstrated in Example 9 and Table3, where the individual areas of cancer tissue, normal epithelia tissue,and stroma, were distinguished by sharply different gene expressionprofiles.

DETAILED DESCRIPTION OF THE INVENTION

Ligation Assays, Generally

A typical ligation assay is illustrated schematically in FIG. 1, whichis discussed in more detail in Example 1. A sample that may containtarget sequences is contacted with a pool of detector oligonucleotideprobes (“probes” or “detectors”). For each target sequence, a pair ofdetectors is provided: a downstream detector (DD) and an upstreamdetector (UD). A downstream detector can have a portion (DR′) that iscomplementary to a region of the target sequence designated as adownstream region (DR). An upstream detector can have a portion (UR′)that is complementary to a region of the target sequence designated asthe upstream region (UR). Here, the terms “downstream” and “upstream”are used relative to the 5′-to-3′ direction of transcription when thetarget sequence is a portion of an mRNA, and for convenience the regionsdesignated as upstream are often shown underlined.

As shown in FIG. 1, the DR′ of the DD and the UR′ of the UD for eachtarget sequence are allowed to hybridize to the corresponding DR and URof the target sequence, if present in the sample. When the DR and UR ofa target sequence are adjacent and the DR′ and UR′ of the pair ofdetector oligos are specifically hybridized to the target sequence toform a hybridization complex, the adjacent detectors DD and UD can beligated. Thus, formation of a DD—UD ligation product serves as evidencethat the target sequence (DR—UR) was present in the sample. In caseswhere the DR and UR of a target sequence are separated by at least onenucleotide, the ligation step can be preceded by (b0) extending the DR′using the sample as a template so the extended DR′ and UR′ becomeadjacent and can be ligated. The ligation product can then be detectedby a variety of means; if desired, the products can be amplified priorto detection. Various detection TempO-Seq™ methods are disclosed herein.

Samples

The samples used in the method can be any substance where it is desiredto detect whether a target sequence of a nucleic acid of interest ispresent. Such substances are typically biological in origin, but can befrom artificially created or environmental samples. Biological samplescan be from living or dead animals, plants, yeast and othermicroorganisms, prokaryotes, or cell lines thereof. Particular examplesof animals include human, primates, dog, rat, mouse, zebrafish, fruitflies (such as Drosophila melanogaster), various worms (such asCaenorhabditis elegans) and any other animals studied in laboratories oras animal models of disease. The samples can be in the form of wholeorganisms or systems, tissue samples, cell samples, subcellularorganelles or processes, or samples that are cell-free, including butnot limited to solids, fluids, exosomes and other particles. Particularexamples are cancer cells, induced pluripotent stem cells (iPSCs),primary hepatocytes, and lymphocytes and subpopulations thereof. Thesamples can be provided in liquid phase, such as cell-free homogenatesor liquid media from tissue cultures, or nonadherent cells insuspension, tissue fragments or homogenates, or in solid phase, such aswhen the sample is mounted on a slide or in the form of formalin-fixedparaffin-embedded (FFPE) tissue or cells, as a fixed sample of any type,or when cells are grown on or in a surface, as long as detectors can beput into contact for potential hybridization with the sample nucleicacids. An optional step in the methods of the invention isdeparaffinization, especially for FFPE samples.

Nucleic Acids

The nucleic acids of interest to be detected in samples include thegenome, transcriptome, and other functional sets of nucleic acids, andsubsets and fractions thereof. The nucleic acids of interest can be DNA,such as nuclear or mitochondrial DNA, or cDNA that is reversetranscribed from RNA. The sequence of interest can also be from RNA,such as mRNA, rRNA, tRNA, siRNAs (e.g., small interfering RNAs, smallinhibitory RNAs, and synthetic inhibitory RNAs), antisense RNAs,circular RNAs, or long noncoding RNAs, circular RNA, or modified RNA,and can include unnatural or nonnaturally occurring bases. The nucleicacids can include modified bases, such as by methylation, and the assayis designed to detect such modifications. The nucleic acid of interestcan be a microRNA (miRNA) at any stage of processing, such as a primarymicroRNA (pri-miRNA), precursor microRNA (pre-miRNA), a hairpin-formingmicroRNA variant (miRNA*), or a mature miRNA.

Relatively short nucleic acids of interest, such as mature miRNAs, canbe lengthened to enhance hybridization to the detectors. For example,many microRNAs are phosphorylated at one end, and can be lengthened bychemical or enzymatic ligation with a supplementary oligo. Thesupplemental oligo can be single-stranded, double-stranded, or partiallydouble-stranded, depending on the ligation method to be used. Ifdesired, the supplemental oligo can be unique to each target sequence,or can be generic to some or all of the target sequences being ligated.The detectors can then be designed with extended DR′ and/or UR′ regionsthat include a portion that hybridizes to the supplemental sequence. Atarget sequence can also be supplemented by adding nucleotides, such asby polyadenylation, where the extended detectors include at least aportion to hybridize to the supplemental polyA tail.

The amount of nucleic acid in the sample will vary on the type ofsample, the complexity, and relative purity of the sample. Because ofthe sensitivity of the assay, the sample can be taken from a smallnumber of cells, for example from fewer than 100,000, 10,000, 1000, 100,50, 20, 10, 5, or even from a single cell or a subcellular portion of acell. The total amount of nucleic acid in the sample can also be quitesmall: less than 100, 50, 20, 10, 5, 2, 1 micrograms, 500, 200, 100, 50,20, 10, 5, 2, 1, 0.5, 0.2, 0.1 nanogram, 50, 20, 10, 5, 2, 1 picogram orless of nucleic acid (see FIG. 6d ), or less than 10, 1, 0.1, 0.01,0.001 picograms of nucleic acid, or amount of a lysate containingequivalent amounts of nucleic acid. The copy number of a particulartarget sequence can be less than 100,000, 10,000, 1000, 100, 50, 20, 10,5, or even a single copy present in the sample, particularly whencoupled with representative amplification of the ligation product fordetection. The amount of input nucleic acid will also vary, of course,depending on the complexity of the sample and the number of targetsequences to be detected.

Selection of Target Sequences for Design of Detectors

The target sequences can be selected from any combination of sequencesor subsequences in the genome or transcriptome of a species or anenvironment, or modified nucleic acids or nucleic acid mimics to whichthe detector oligos can bind or hybridize. The set can be specific for asample type, such as a cell or tissue type. For some sample types, thenumber of target sequences can range in any combination of upper andlower limits of 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000,10,000, 20,000, 23,000, 30,000, 38,000, 40,000, 50,000, or more. Thenumber of target sequences can also be expressed as a percentage of thetotal number of a defined set of sequences, such as the RNAs in thehuman transcriptome or genes in the human genome, ranging in anycombination of upper and lower limits of 0.1%, 0.2%, 0.5%, 1%, 2%, 5%,10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 65%, 60%, 70%, 75%, 80%, 85%,90%, 95%, and 100%. Where large sets of detector oligos are used, it canbe useful to check the full sequence of each oligo for potentialcross-hybridization to other oligos in the set, where, for example, oneoligo may inadvertently serve as an template to other detectors. Whilesuch non-specific artifacts can be identified by sequence, and aretypically discarded from detection results, they may representnoninformative hybridization events that compete for reaction resources.

The target sequence of interest can be a subsequence of anycancer-associated marker, such as any of genes listed in Tables 1, 2,and 3.

Detector Oligonucleotides

Based on the particular target sequences, the invention provides poolsof detector oligos where a target sequence has a pair of upstream anddownstream detectors (UD and DD) that correspond to DR and UR, which aretypically subsequences of the entire nucleic acid sequence of interest.Detector oligos can be designed to hybridize to the target sequence so asingle-stranded sequence portion of the target sequence remains betweenthe detectors, which can then be filled in, such as by reversetranscriptase or polymerase, thereby extending a detector to bring iteffectively together with the other detector so they can be ligated.

Detectors can be provided to detect targets that contain mutationsincluding individual single-nucleotide polymorphisms (SNPs), genefusions, and exon-splicing variants, or modifications such aspseudouridylation and methylation. For example, DNA samples of interestcan have bases that are methylated, such as N⁶-methyladenine (m⁶A). DNAfrom mammals and other species can have one or more 5-methylcytosine(m⁵C) modified bases, often appearing in GC, CHH and CpG dinucleotides,which sometimes form CpG-rich islands. For RNA samples, modifications tobe detected by the invention include methylated ribonucleotides havingm⁶A (often playing a role in mRNA regulation), m⁵C, andN¹-methyladenosine (m¹A), which can be dynamically modified in mRNAs andis sometimes correlated with protein translation.

Detectors can contain blocking groups, modified linkages between bases,unnatural or nonnaturally occurring bases or other unnatural ornonnaturally occurring components. An individual target sequence canhave more than one set of DRs and URs, which can be selected by the userto optimize the performance of the assay. Multiple sets of DRs and URscan provide multiple measurements of the same target sequence or ofdifferent portions of the target sequence, such as different exons orexon junctions, or provide measurement of a portion of sequence that isnot mutated versus a portion of sequence that may harbor a mutation.

The detector oligos themselves can be DNA, RNA, or a mixture or hybridof both. If desired, they can have a modified nucleotide such as dideoxynucleotides, deoxyUridine (dU), 5-methylCytosine (5mC),5-hydroxymethylCytosine (5hmC), 5-formylCytosine (5fC),5-carboxylCytosine (5caC), and Inosine. Yet other modifications todetector oligos include modified bases such as 2,6-diaminopurine,2-aminopurine, 2-fluro bases, 5-bromoUracil, or 5-nitroindole. Otherdetector oligos can have a modified sugar-phosphate backbone at one ormore positions. Such modifications include a 3′-3′ or 5′-5′ linkageinversion, a locked nucleic acid (LNA), or a peptide nucleic acid (PNA)backbone. LNAs can be useful for their stronger hybridization propertiesto complementary bases, enhancing the selectivity or the overall bindingaffinity for the detector oligo as a whole. The modified bases or bondscan also be used at positions 1, 2, or 3 away from the point ofligation.

As shown schematically in FIG. 1, a downstream detector (DD) has acomplementary downstream region (DR′), which can be at least 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 35, 40, 45, or 50nucleotides in length. Similarly, an upstream detector (UD) has acomplementary upstream region (UR′), which can be at least 4, 6, 8, 10,12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 35, 40, 45, or 50 nucleotides inlength. In a given pair of DD and UD for a target sequence, the DR′ andUR′ need not be exactly the same length, but will typically be similarso they can hybridize to the target under similar conditions andstringency.

As discussed in more detail below, the detectors can be optimized forligation, such as by providing a 5′-phosphate on the UD, although thisis not necessary, depending on the selection of ligase or other ligationmethods. Ribonucleotides can also be substituted at the ligatable endsof the DD and UD to increase the specificity and efficiency of ligation,as when an RNA ligase is used.

Anchored Detectors

In one configuration of the TempO-Seq™ assay, the upstream detector hasa second region (UR2′) that is complementary to a second region of thetarget sequence (UR2), as illustrated in FIG. 2a . Because the tail ofthe UD can hybridize to a separate portion of the target, thisconfiguration can be described as an “anchored” detector, as in FIG. 2b. The anchor at the 3′ end of the UD hybridizes with the target to forma double-strand and is thus configured to resist digestion to nucleasesthat degrade single strands, such as 3′ exonucleases like exo I.

As a separate target-binding region, the anchor UR2′ can be used toprovide additional discrimination between similar sequences, such asisoforms of a family of genes where sequence differences betweenisoforms are found beyond the range of the DR and UR target sequence.The UR2′ can be at least 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 35, 40, 45, or 50 nucleotides in length. The UR2′ can beseparated from the UR′ by a noncomplementary region (CP1), which can beat least 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 35, 40, 45, 50,60, 70, 80, 90, or 100 nucleotides in length. In general, the UR2′ willbe upstream relative to the UR′. If an amplification region (such asP2′) is present, it can be upstream of the UR′, such as within the CP1or part of UR2′ to allow amplification of the UR′ portion as shown inFIG. 2c to generate the amplification products (AP) in FIG. 2 d.

In a mirror-image configuration, it is the downstream detector that hasthe anchor region (DR2′) complementary to a second region of the targetsequence. The DR2′ anchor hybridizes to a DR2 on the target so that theconfiguration resists the action of 5′ ss-exonucleases. The UR2′ of theDD will generally be downstream relative to the UR′. If an amplificationregion (such as P1) is present, it can be downstream of the DR′ to allowamplification of the DR′ after ligation. Anchored DDs and UDs can beused separately or in combination to resist a cocktail of nucleases.

Because the separate anchor region of the detector can affect thehybridization characteristics of the detector via monomolecularkinetics, the compositions and relative lengths of the DR2′, CP1(s),DR′, UR′ and UR2′ can be tuned to optimize target selectivity betweenthe detector pair and among the pairs of the detector pool.

Detectors that are not used in the ligation reaction can be degraded asshown in FIG. 2e . Moreover, incompletely bound detectors, such as thosein FIG. 2f , can also be degraded, for example when the UR′ of a UDbinds to the UR of a target, but the UR2′ does not bind, whether becausethe UR′ is bound to a non-target sequence or to a target that wasrelated to the intended target UR but lacked a UR2. Similarly, ananchored DD that binds a DR2 but not the DR of a target will besusceptible to a 3′ ss-exonuclease (or will not generate a validligation product with a corresponding UD). Other detectors will fail tobe amplified, for example detectors in excess of target sequence in thesample or detectors that are bound nonspecifically to nontargetsequences. The use of anchored detectors can therefore increase thespecificity of the ligation assay for target sequences while allowingnucleases to degrade excess or unused detectors.

Blocked Detectors

Another configuration has detectors that are nuclease-resistant byhaving a nuclease-blocking group at or adjacent to one end. FIG. 2hshows a DD, having a 5′-blocking group, that can be used in combinationwith a 5′ exonuclease. Also shown is a UD having a 3′-blocking group foruse with a 3′ exonuclease. Preferably when a 5′ or 3′ exonuclease isused where there are multiple targets and pairs of detectors, all of thedownstream or upstream detectors have a 5′ or 3′ block, respectively.

Useful configurations for resisting nucleases include termination withan inverted nucleotide such as deoxythymidine (idT), a dideoxynucleotidesuch as dideoxythymidine (ddT or iddT), or 2′/3′-acetyation of theterminal nucleotide. Depending on the substrate preferences of thenuclease selected, one or more of the other modified nucleotidesdescribed earlier can be used as a blocking group. Alternatively, one ormore of the terminal nucleotides are attached to the rest of the oligovia one or more phosphorothioate bonds instead of naturally occurringphosphodiester bonds. Other modifications that may resist a nucleaseinclude the LNA or PNA backbones discussed earlier. In someconfigurations, a hairpin loop or other secondary structure on thedetector can serve as the nuclease-blocking group for a detector. Oneend of the hairpin can have a blocking group. In other configurations,prior to hybridization, a protein or other component can be bound the 5′end of a DD or the 3′ end of a UD, such as a sequence-specificsingle-strand-binding protein like a far upstream element (FUSE) bindingprotein (FUBP) via a ssFUSE sequence incorporated into a detector. Ifthe 5′ end of a DD or the 3′ end of a UD detector is configured to beimmobilized, whether permanently or reversibly, to a solid phase, thesolid phase itself can serve as a block against nuclease activity on thedetector. It can be useful to combine any of the preceding features in asingle detector or both detectors to resist the action of the nucleaseselected and to provide other advantages, such as stability andhybridization properties.

Protectors

Yet another configuration provides one or more oligos that protect thedetectors by hybridizing to the DD or UD at a region that will notinterfere with hybridization of the DR′ or UR′ regions complementary tothe target sequence. For example in FIG. 2i , a DR2 protector oligo isprovided to hybridize to a DR2′ region at the 5′ end of the DD, forminga double-stranded configuration (indicated by a brace) that is resistantto 5′ exonucleases. If a 3′ exonuclease is to be used, then a UR2protector can be provided to form a double-strand at the 3′ end of theUD. The protector oligos can themselves be protected from exonucleaseactivity by a blocking group or bond as described above. For example, a3′-blocked UR2 protector is shown in FIG. 2i , and a 5′-blocked DR2protector is shown in FIG. 2j . If a cocktail of 5′ and 3′ exonucleasesis to be used, then both DR2 and UR2 protectors can be provided,optionally with 5′- or 3′-blocking groups, respectively.

Detector Labels

Where the ligation assay proceeds directly to a detection step, eitheror both detectors can be designed to be labeled appropriately fordetection. For example, the detector can be conjugated to any number ofmolecular or physical entities, labeled with a crosslinker, activatablecrosslinker, activatable cleavage group or enzymatically cleavablegroup, optical, color or fluorescent dye, latex or other beads, quantumdots, or nanodots, or nanoparticles. Any of these entities can also befurther modified or conjugated to other entities. The label can alsotake the form of an additional nucleotide sequence that serves to enabledetection and identification, such as a barcode sequence. For example, auseful barcode sequence can uniquely identify the specific gene ortarget sequence, or a group of select genes or target sequences withinthe sample that are being measured. Such sequences can be positionedbetween the UR′ and P2′ sequence, and/or between the DR′ and P1sequence, so they are amplified when using flanking primers. Thissequence can also be a random sequence, useful for identifying thenumber of copies of the target gene in the sample, independent of theparticular efficiency of any amplification step.

Cleavable Detectors

It can be desirable for a detector oligo to contain one or othermodifications that can be selectively cleaved by treatment after theligation or optional amplification step. For example, a detector oligocan have a dU located so that it will not interfere with hybridizationor ligation steps. After ligation, however, products incorporating thedU oligo can then be cleaved by dU-specific enzymes, such as uracil-DNAglycosylase followed by endonuclease VIII. Another selectively cleavablesite can be a restriction enzyme cleavage site that is not present inthe target sequences to be detected. Yet another cleavage site is aphotocleavable site. It may also be useful to incorporate a moiety thatcan be crosslinked before or after ligation, such as a photoactivatableor chemically activatable crosslinker.

Multiple Detectors for a Gene

Multiple detector oligo (DO) sets targeting different sequences within agene can be designed and synthesized for use to detect that gene. EachDO set hybridizes to its targeted sequence independently of thehybridization of other DO sets to each of their respective targetedsequences. Thus, the statistical reliability, statistical power, ofmeasurement of the gene itself can be increased by use of multiple DOset targeting that gene. Measurement CVs can be reduced. Furthermore, ifsecondary structure, protein binding, or other factor modulates thehybridization of one DO set, and thus affects resulting measure of geneabundance by that DO set, then the counts from other DOs unaffected bysuch factors can be used to provide more accurate measure of geneabundance. Outlier analysis can be used to identify such deviations ofDO set measurements. In the case that the expression of a gene is lowabundant, or that the amount of sample is small, such as from a singlecell, and thus the number of gene molecules is low, hybridization of aspecific DO set to that low amount of gene may not be sufficient toprovide an amplifiable ligated product every time across repeat samples,and hence, not produce sequencing counts from some samples. The use ofadditional DO sets targeting other sequences within the same geneincreases the probability that some of those DO sets will produce countsif the gene is actually expressed, and thus use of multiple DO sets canbe used to increase the sensitivity of measurement of low expressed, orlow numbers of gene molecules in a sample. The no sample backgroundcounts can be used to validate that DO counts result from the presenceof the gene even though not all DO sets produce counts. The concurrenceof more than one DO set reporting the presence of the gene can be usedas a measure to validate that the DO counts result from the presence ofthe gene even though not all DO sets produce counts. Because the DO setshave a defined sequence, each DO set measurement represents independentmeasurements of defined target sequences, permitting statistical methodsto be applied to determine that a gene is expressed or present in thesample or not.

Detecting Modified Nucleotides

In a particular embodiment, multiple detectors can be used to detect thepresence or absence of modifications to a nucleic acid. For example, afirst pair of detectors can be directed to a first target sequence of afull-length nucleic acid of interest, such as an mRNA, where the firsttarget sequence is suspected of having a modification, such asmethylation, at a particular position for interrogation. The first pairof detectors may yield one detection result (e.g. generation of ananalytical ligation product or amplicon) when the modification ispresent at the position, and yield a different detection result (e.g. noanalytical product) when the modification is absent from the sameposition. Detectors, which are directed to one or more different targetsequences or positions of the full-length nucleic acid, can be used as apositive control for the presence of the full-length nucleic acid.

Hybridization

Returning to the steps of the assay, the detectors are provided so thatthey contact the sample to allow the detectors to hybridize specificallyto the target nucleic acids. Hybridization conditions can be selected bythe skilled artisan to allow and optimize for hybridization between thepolynucleotides with the desired degree of specificity or mismatches,and such conditions will vary with the lengths and compositions ofsequences present in the hybridization reaction, the nature of anymodifications, as well as conditions such as the concentrations of thepolynucleotides and ionic strength. Particular hybridizationtemperatures include 30°, 32.5°, 35°, 37.5°, 40°, 42.5°, 45°, 47.5°,50°, 52.5°, 55°, 57.5°, 60°, 62.5°, 65°, 67.5°, 70°, 72.5°, 75°, 77.5°,80°, 82.5°, 85°, 87.5°, and/or 90°. Particular hybridizationtemperatures can be achieved by ramping the temperature up or down atvarious rates and profiles, such as timed temperature plateaus, one ormore incremental increases or decreases of 5° C., 10° C., or 15° C., andrepeated cycling between two or more temperatures. Ions such as Li⁺,Na⁺, K⁺, Ca²⁺, Mg²⁺ and/or Mn²⁺ can also be present from 0, 1, 2, 5, 10,20, 50, 100, 200, and 500 mM, and such ions can affect the selection ofthe other hybridization conditions. Hybridization is also affected bysteric crowding components such as branched polysaccharides, glycerol,and polyethylene glycol. Further additives can be present in thehybridization (and subsequent) reactions, such as DMSO, non-ionicdetergents, betaine, ethylene glycol, 1,2-propanediol, formamide,tetramethyl ammonium chloride (TMAC), and/or proteins such as bovineserum albumin (BSA), according to the desired specificity.

Optionally, the conditions for hybridization can be adjusted orfine-tuned to permit other steps to be performed in the sameenvironment. For example, the same buffers used for hybridization can beused for lysing cells in a sample, promoting hybridization of certaincell types, facilitating removal or permeation of cell walls, cellmembranes, or subcellular fractions, as desired. Depending on theligation method used in the assay, hybridization conditions can beselected to be compatible with conditions for ligation as is, or withthe addition of one or more components and preferably without requiringa change of the reaction container when transitioning from hybridizationto ligation steps.

Ligation

The ligation reaction can occur by chemical ligation or by using aligase enzyme or a ligation-facilitating co-factor. A variety ofnick-repairing ligases are commercially available to catalyze theformation of a phosphodiester bond between adjacent single-strandedpolynucleotides when hybridized to another single-stranded template,such as to join DNA to RNA when hybridized to template. An example isbacteriophage T4 DNA ligase, which is generally understood to use ATP asa co-factor. The ATP can be supplied during the ligase reaction. Inother reactions, the ligase can be pre-adenylated. In yet otherreactions, the UD must be pre-adenylated at the 5′ end, as with a 5′ AppDNA/RNA ligase. The UD in a typical reaction will have a 5′-phosphate tofacilitate ligation to the DD, although this is not necessary, dependingon the selection of ligase and ligation conditions. (Where a5′-phosphate on the DD is required for efficient ligation, using acomparable oligonucleotide without 5′-phosphorylation can be used toinhibit or reduce undesired ligation.) Preferred ligation conditionsinclude 10, 25, 50, 100 mM Tris-HCl (pH 7.5, 8.0, or 8.5); at least 10mM, 5 mM, 2 mM, 1 mM MgCl₂; at least or at most 2 mM, 1 mM, 0.7 mM, 0.5mM, 0.2 mM, 0.1 mM, 0.05 mM, 0.02 mM, 0.01 mM, 0.005 mM, 0.002 mM, or0.001 mM ATP; or at least 10 mM, 7 mM, 5 mM, 2 mM, 1 mM, 0.5 mM DTT orother antioxidant. T3 DNA ligase can also be used, which can ligate abroader range of substrates and has a wider tolerance for saltconcentration. As with other steps, the temperature can be selectedaccording to the characteristics of the reaction components andconditions such as ionic strength.

As discussed above, the ligation step can be preceded by an optionalextension step, as in FIG. 1, step (b0). The ligation step can also bepreceded by an optional cleavage step, such as by a nuclease, to removeany overhangs. In other cases, a portion of the DD can overlap with theUR sequence to which the UD hybridizes, so that after hybridization ofthe UD and the DD, there is an overhang sequence of 1, 2, 3, or morebases. A useful enzyme for removing an overhang is a Flap endonuclease,such as Fen-1, which cleavage leaves a ligatable 5′-phosphate.

Amplification

If desired, the ligation product can be amplified (for example by PCR orqPCR) to facilitate detection. Amplification methods and instruments arecommercially available, including PCR plate and droplet formats, and theamplification enzymes (such as Taq and its commercial variants) andreaction conditions can be selected and tailored to the particularplatform. Optionally, the polymerase selected for amplification can havestrand-displacing activity. As illustrated in FIG. 1, the detectors canhave additional sequences (“tails”) including primer hybridizationsequences (e.g. P1, P2′) or complements thereof, that serve asamplification sequences, so that after ligation, the ligation productcan be amplified with a pair of amplification primers (P1, P2).

Amplification can also be linear, or achieved by any number of methodsother than PCR. If desired, the amplification primer can incorporate abarcode sequence, for example a barcode sequence that uniquelyidentifies the sample in a multi-sample experiment, and optionally hasredundant and/or error-correction features. In some experiments, forexample, different sample barcodes can be used for 96, 384, 1536, ormore generally 2^(n) or 4^(n) different samples that are prepared withdifferent barcodes separately for some steps, such as hybridization,ligation, and amplification, and combined for others, such as detection.The barcode sequence can be incorporated into the primer, such as 3′ tothe amplification sequence, so that the barcode becomes part of theamplified strand. In other instances, the amplification sequence of theprimer can be extended by an additional sequence to provide a primerhybridization sequence that can be used for use in subsequent sequencingsteps. The barcode may also be interposed between the amplificationsequence, and if desired, the extended amplification sequence, andanother sequence that can be used for capture, such as capture onto asurface as part of a sequencing process, and/or for yet another primerhybridization sequence that is used for sequencing. In each case thebarcode will be amplified with the rest of the detector sequences, forinstance forming a single amplified, elongated molecule that containssequencing primer hybridization sequences, sample barcode, and agene-specific sequence, which may include a gene-specific barcode or atarget molecule-specific barcode as well as sequence or complement tothe sequence of the target gene. In the case where the targeted oligo isa cDNA, a gene-specific sequence or a sample-specific sequence can beadded as part of the primer used for reverse transcription, and be apart of the sequence targeted by the UD and DD.

In other instances, methods known in the art can be used to amplify theligated DD and UD sequences, such as by repetitive cycles of (1)ligation, (2) heating to melt off the ligated product, (3) cooling topermit hybridization of DD and UD to the target, (4) ligation, thenrepeating the heating (2), cooling (3), and ligation (4) steps. Theseadditional amplification steps can be performed before amplificationstep (c), during which the sample barcodes and other sequences are addedto the ligated UD and DD sequence. The target of the UD and DDhybridization may also be amplified by whole transcriptome amplificationof RNA or amplification of cDNA.

Detection

The ligation product (or its amplicons) can optionally be detected bymethods such as sequencing, qPCR, end point PCR, enzymatic, optical, orlabeling for detection on an array or other molecule detection. Otherdetection methods include flow-through systems for counting labeledmolecules. Depending on the detection method, the skilled user will beable to modify the design of the detectors and amplification primers toinclude functional features that are appropriate, such as for bridgeamplification on a sequencing flow cell. The experimental resources usedfor amplification and detection can be limited and are often among themost expensive, and their consumption can be optimized by reducing thenumber of non-informative assay components present at various stages ofthe assay.

Nucleases

Accordingly, the invention provides optional nucleases and assaycomponents that are configured to resist degradation to enable moreefficient use of resources and more sensitive detection. As a furtheradvantage, the invention enables a simpler assay workflow that can beperformed in a single reaction container or entirely in liquid phase.

The nuclease can be an enzyme that digests or degrades single strands ofnucleic acids. Preferably the nuclease does not digest (or hassignificantly less activity on) double strands, including DNA:RNAhybrids. For example, the nuclease can have less than 10%, 5%, 2%, 1%,0.5%, 0.2%, or 0.1% the activity on double strands compared tosingle-strands on a molar substrate ratio under the same conditions.Similarly, the nuclease can be selected so it does not appreciablydigest at single-stranded nicks in a double-strand. The nuclease can bean endonuclease that degrades single strands, such as mung bean nucleaseunder certain conditions. The nuclease can also be an exonuclease thatdegrades single strands, which can be single strands of DNA. Forexample, a nuclease having single-stranded 3′-to-5′ exonuclease (3′ exo)activity includes Exonuclease I from E. coli (exo I) and T3 exonuclease.Enzymes such as exonuclease T (RNase T), which has 3′ exo activity onDNA and RNA single strands, can be used as long as the detectors havebeen ligated and the RNA strands are no longer needed in the assay.Nucleases having single-stranded 5′-to-3′ exonuclease activity includeexonuclease VIII and RecJ_(f). The nuclease can be an enzyme thatdigests 5′ overhangs or flaps, such as Flap endonuclease 1. Nucleasescan be used singly or in a cocktail of nucleases, such as a pair of 3′and 5′ exonucleases.

The nucleases can be used at various stages of the assay. For example, anuclease can be provided (b2) after the ligation step (b1) to removeunligated or excess detectors, as in FIG. 2e . The nuclease can alsodegrade detectors that are only partially or nonspecifically hybridizedto target sequences, as in FIG. 2f . If compatible with the ligationconditions used, the nuclease can also be provided during the ligationstep (b1 and b2 together), or even before the ligation step (b2, thenb1) as long as it does not interfere with the intended detection oftarget sequences. Depending on the assay design, the nuclease can beprovided before, during, or after the optional (b0) extension and (d)amplification steps, or at multiple steps to effect the desired purposeof removing undesired target, detectors, other oligos, or any products.

When the nuclease activity is no longer desired, the nucleases can beremoved or inactivated, such as after the ligation step. Nucleases canbe inactivated by methods selected for a particular nuclease but willnot substantially interfere with the rest of the assay. For somenucleases, a nuclease inhibitor (as in FIG. 4, lower right) or chelatingagent, such as EDTA, can be added as long as it does not interfere with(or can be removed prior to) a subsequent step that may require Mg⁺⁺ forexample. Other nucleases can be inactivated by heat, for example singleor repeated incubation at 70° C., 75° C., 80° C., 85° C., 90° C., 95° C.or 98° C., for 1, 2, 5, 10, 15, 20, 25, 30, 45 minutes, or 1 hour. Ifmore than one nuclease is used, either or both may be inactivatedindividually or by the same means. To resist the activity of nucleasesprovided at one or more steps of the invention, components of the assayare provided by the invention in various configurations that permitdetection of target sequences. Selection of the configuration methodwill depend, of course, on the particular nuclease being used.

Steps in Solid and/or Liquid Phases

In other embodiments, one or more of the steps can be performed inliquid phase, such as in a microfluidic system, so that one or more ofthe steps does not involve capture to a solid phase, such as to a beador a plate surface. For example, any one or combination of thehybridization, extension, ligation, nuclease digestion, amplification,or detection steps can be performed in liquid phase.

In some embodiments, the sample is provided in a solid phase, such as anFFPE, so that it remains in solid phase for one or more steps of thedetection process. When in solid phase, the sample can be washed betweensteps to remove unused assay components or to reduce background, forexample after hybridization or after ligation.

In a mixed phase assay, a solid phase can be used to immobilize one ormore of the sample, the detector oligos, the hybridization complex, theextension product, the ligation product, or the amplification product.In particular, the target nucleic acid can be attached to a solidsurface during the hybridization step, the ligation step, or both. Thesolid surface can be a bead, such as a magnetic, nonmagnetic, polymeric,reversible immobilization, or latex bead, or compound beads thereof, ora relatively flat surface such as a plate or flowcell surface,optionally with coatings of similar materials. The mixed phase formatallows the components to be transferred from one reaction environment toanother, or the conditions to be changed as the components remain in onecontainer.

Adding Successively to the Same Reaction Container

Alternatively, the reactions can be optimized so that at least one ofsteps is performed by adding reagent, such as an enzyme or buffercomponent, successively, so that a reaction takes place in the samecontainer as the preceding step, optionally without requiring anintervening wash or transfer step. Preferably, the sequence of additionsdoes not require significant additions of liquid volumes to dilute thecomponents for the next reaction, for example no more than 1-, 1.5-, 2-,2.5-, 3-, 5-, 10-, 15-, or 20-fold dilution between the initial sampleand preparation for detection. The components to be added can beprovided in a kit, as described below.

Steps in Situ; Cross-Linking, Photocleavage, Elution

In some embodiments, the hybridization, ligation, or extension steps canbe performed while the target sequence is in situ, as with FFPE samples.This can be particularly useful, for example, when the sample is on ahistological slide, so that the ligation is known to occur at arecordable location and can be compared to similar reactions at otherlocations on the slide. It useful for any sample where the targetsequence is part of a nucleic acid is fixed to the tissue. The ligatedprobes can remain at the location while other steps are performed, suchas imaging or detection of other analytes at or near the location. Theseother analytes can be any of the nucleic acids described herein,including modified nucleotides, carbohydrates or lectins, proteins andother antigens, and any other stainable molecule or feature that can bevisualized. These other analytes in situ can be present on the surfaceof the sample, treated to expose them on the surface, or be madeaccessible to reagents such as stains to aid their visualization, suchas by permeabilization.

If desired, the ligated probes can remain in situ more securely by avariety of chemical or enzymatic methods for cross-linking to the site,which can be permanent or reversible, such as by a photocleavable linkas with using a cyanovinylcarbazole nucleoside analog (^(CNV)K). Thearea to be photocleaved can be any shape or size, and can be focused onone or a few selected cells of interest, or can focus on a histologicalor pathological feature. The photocleavage steps may also be performedwhether the sample is wet or dry.

In a particular embodiment, the ligation products can be eluted from thesample in situ for collection and further processing, preferably elutingfrom small areas to preserve the location information and morphologicalcontext of the ligation reaction products. Elution can simply be by heatin low salt, effected by the PCR process, or by addition of base. Theeluted area can be smaller than 2 mm², 1, 0.5, 0.2, 0.1, 0.05, 0.02,0.01, 0.005, 0.002, 0.001 mm² (1000 μm²), 500 μm², which covers therange of single human cells of many types.

Photocleavage and elution steps can be coordinated so a first area isphotocleaved, followed by elution of a second area. The first and secondareas can be coextensive, overlap, or be larger or smaller relative toeach other. In other combinations, an area may first be nonspecificallywashed or selectively eluted for some components, then photocleaved,followed by elution and collection of other components.

In a particular embodiment, samples are dried, fixed, optionallypermeabilized, and optionally processed prior to or during the assay. Inyet another embodiment, samples are simply preserved by fixation beforethe assay.

TempO-Seq™ Assays

Standard Version

A “standard” version of the TempO-Seq™ assay provides a method fordetecting target nucleic acid sequences in a sample, wherein a targetsequence has a downstream region (DR) and an upstream region (UR). Thesteps include (a) contacting the sample with a pair of detector oligos.The detector pair comprises a downstream detector oligo (DD) having acomplementary downstream region (DR′) and a separate upstream detectoroligo (UD) having a complementary upstream region (UR′). At least one ofthe DD or UD has a second complementary region (DR2′ or UR2′) separatedfrom the DR′ or UR′ by a noncomplementary region (CP1) that does nothybridize to the target nucleic acid. Thus, the DR2′ or UR2′ canspecifically hybridize to a DR2 or UR2 of the target nucleic acid. Thisallows the pair of detectors to hybridize specifically to the targetnucleic acids. The method continues by (b1) ligating the DR′ and UR′ ifboth are specifically hybridized to the DR and UR of a target sequence;and (b2) exposing hybridization complexes to at least one nuclease thatdegrades single strands but does not significantly degrade doublestrands. Thus, nonspecifically hybridized DDs and UDs are degraded bythe nuclease. The ligation product serves as an analytical product thatindicates the presence of the target sequence in the sample.

In a particular embodiment, the assay targets 50 nucleotide regions inRNAs with pairs of detector oligos (DOs), which share universal PCRprimer landing sites. After annealing, the adjacent DOs are ligatedtogether and amplified by PCR (which can also add sample tag sequencesand sequencing adapters). A single PCR can primer pair amplify allligated probes in a single sample. Attaching unique tag sequences thatare sample-specific can allow sample pooling into a sequencing libraryof 384 or more samples per flow cell.

As disclosed above, the sample can be a tissue sample, can be mounted ona slide, or can be an FFPE. The target nucleic acid can be from an FFPEsample, or can be in situ. The standard version can have a step ofeluting the ligation product.

The standard assay can be performed with FFPE samples, as discussed inExample 5 and illustrated in FIG. 4. The TempO-Seq™ assay iscommercially available as a kit in a Whole Transcriptome version(BioSpyder Technologies, Inc., Carlsbad, Calif.).

Modified Version of TempO-Seq™ Assay

A “modified” version of the assay is described in Example 3 andillustrated in FIG. 3.

in Situ Version of TempO-Seq™ Assay

An in situ version of the assay is described in Example 5 andillustrated in FIG. 6. In this version, probes that are not bound to thesample can be washed away, reducing assay background, and increasingspecificity and overall sensitivity. The method can detect a nucleicacid sequence from a selected area of a sample in situ, by performing inany order: imaging the sample for the presence or absence of an analyte;selecting an area of the sample based on the imaging; detecting a targetnucleic acid sequence by any of the detection methods for nucleic acidsequences herein; and collecting the ligation products from the selectedarea for analysis.

The selected area can be a morphological feature, which can bevisualized by one or more stains. Any histologic stain can be used toimage the sample. Useful stains include fluorescent dyes, enzymes (suchas peroxidase or alkaline phosphatase), as well as radioactive labels.Immunostaining or other antibody-based staining methods can be used,including immunohistochemical staining of tissue sections.

The analytes can be any of the nucleic acids or modified versionsdescribed herein. More generally, the analytes can be any detectablemolecule such as proteins, carbohydrates, or their binding partners orstain components.

The detection of many antigens can be improved by antigen retrievalmethods that break some of the protein cross-links that may have formduring fixation, thereby uncovering previously hidden antigenic sites.Retrieval methods include heating, such as heat-induced epitoperetrieval (HIER) and using enzyme digestion, such as proteolytic inducedepitope retrieval (PIER).

Individual steps in this version can be automated or performed manually,or using any slide-staining apparatus where temperature can becontrolled during incubations.

Kits

The invention provides kits for performing the methods described above,comprising detector oligos, and optionally a nuclease, a ligase, and/ora polymerase. The kits can further provide reaction buffers for theenzymes in the kit or buffer components to be added to reactionssuitable for the enzymes. The component can be suitable for addition toa container for an enzyme reaction to prepare a suitable reaction bufferfor the enzyme. The component can also be selected to be compatible withthe reaction buffer for the preceding step of the method so that thecomponent can be added to the same container to form a reaction bufferfor the next enzyme to be used. Thus, the components can be selected toenable an “add-add-add” strategy for multiple steps of the assay tominimize transfers of sample, oligos, enzymes and/or solutions betweenseparate containers.

The kits can also have eluent solutions suitable for removingoligonucleotides, such as ligated oligonucleotides, from a tissue samplefor further analysis. The kits can further have amplification primerssuitable for use with the detectors of the kit.

As disclosed above, the kit can have a pair of detector oligos, whichpair comprises a downstream detector oligo (DD) having a complementarydownstream region (DR′) and a separate upstream detector oligo (UD)having a complementary upstream region (UR), wherein at least one of thedownstream detector (DD) or the upstream detector (UD) has a secondcomplementary region (DR2′ or UR2′) separated from the DR′ or UR′ by anoncomplementary region (CP1) that does not hybridize to the targetnucleic acid and that has an amplification region (P1 or P2′), wherebythe DR2′ or UR2′ can specifically hybridize to a DR2 or UR2 of thetarget nucleic acid, and at least one nuclease. Kits can also includeone or more eluent solutions to remove oligos, such as unligateddetectors, or in a separate step, to elute ligation products from thetissue sample.

The kits can also contain a stain, such as a histological stain, such ashemotoxylin or eosin. The stain can also have an antibody, such as forimmunostaining, for detecting an analyte in the sample, as describedherein.

Diagnostic and Other Methods

The present invention provides a method for detecting a neoplastic stateof a cell by detecting one or more cancer marker sequence in a cell. Asshown in Table 3 below, in a selected are, ligation products of a secondcancer marker sequence can be detected in significantly fewer numbers,such as less than 0.1%, 0.05%, 0.02%, 0.01% or 0.005% than the firstcancer marker sequence.

The invention provides methods for generating a gene expression profilefor a selected area for a plurality of target sequences.

The invention also provides methods for detecting a neoplastic state ofa cells in a tissue detecting a plurality cancer marker sequences oncells in two separate areas of the tissue.

The invention further provides methods for diagnosing a disease statewherein the target sequences are detected in the area of a morphologicalfeature.

Instruments

The invention provides instruments, which can be automated, for imagingsamples such as FFPEs or slides, selecting focal areas, and eluting torecover analytes from those areas. The instrument can have an imagingcomponent, a component for collecting ligation products from theselected area, and a component for transferring the products to anexternal container.

An example of the instruments of the invention is the CellSensus™digital molecular pathology platform. This platform combines a digitalimager for slides, and a mechanism for automatically recovering probesfrom selected areas, and transferring them, for example to PCR tubes.The platform also includes software to control some or all of thesefunctions and perform analysis.

EXAMPLES Example 1 Representative Ligation Assay

A representative method is provided to illustrate ligation assays. Here,over 100 RNA expression products were detected in a sample of cellsusing a multiplex assay format. For each expression product, the assaywas designed to detect one or more target sequences within the fullsequence of the product. For example, in human cells, a GAPDH gene ofinterest encodes the enzyme glyceraldehyde 3-phosphate dehydrogenase;three different portions within the RNA transcript of the GAPDH genewere independently detected as target sequences. One such RNA targetsequence, identified here as GAPDH_2, was where a 5′ end was designated“upstream” (underlined) and the 3′ end was designated “downstream” forthe direction of transcription and translation. A downstream region (DR)was defined as the downstream 25 bases of GAPDH_2, which has acomplementary DNA sequence of DR′. The upstream region (UR) was definedas the upstream 25 bases of GAPDH_2, which has a complementary DNAsequence of UR′.

For GAPDH_2, a pair of detectors was designed: a downstream detector(DD) having the DR′ sequence, and an upstream detector (UD) having theUR′ sequence. Similar pairs were designed for each of the targetsequences to provide a pool of detectors for the assay. In this example,all the upstream detectors were phosphorylated at the 5′ end.

In this particular example, an amplification step was to be performedlater in the experiment using two primers, P1 and P2, so all UDs in theexperiment included a primer sequence (P1) and all URs included acomplementary primer sequence (P2′). Because amplification is notnecessary to the practice of the invention, however, the sequence of thespecific primers and primer sequences is a matter of selection to suitthe particular amplification method, if used.

At least 10 ng of RNA isolated from human kidney or liver cell lines wasplaced in a well of a microtiter plate for each assay experiment. Toeach well was added 20 μL of 2× Binding Cocktail, which contained 5 nMof each detector (providing a final input of 0.1 pmoles per oligo), 100nM biotinylated oligo(dT)₂₅, and 5 μL, streptavidin-coated magneticbeads in a Wash Buffer (40 mM Tris-Cl pH 7.6, 1 M NaCl, 2 mM EDTAdisodium, 0.2% SDS).

The plate was heated for 10 min at 65° C. to denature the RNA, then thetemperature was ramped down over 40 min to 45° C. to allow the detectorsto anneal to the target sequences in the RNA sample. The plate was thentransferred to a magnetic base to immobilize the beads, allowing thesupernatant, containing unbound and excess detectors, to be aspiratedfrom the wells. The beads were washed at least three times with 50 μLWash Buffer.

To each well was added 5 Weiss units of T4 DNA ligase in 20 μL, of 1×ligation buffer, as provided by the supplier. After the beads wereresuspended by pipette, the plates were incubated for 60 min at 37° C.to allow target-dependent ligation of DDs to UDs as appropriate.

After the ligation reaction, the beads were immobilized and washed twicewith 50 μL Wash Buffer. To release the ligated detectors from their RNAtargets, the beads were resuspended in 30 μL and incubated for 5 min at65° C. After incubation, the beads were immobilized, and the supernatantwas removed and transferred to a storage plate.

For the optional amplification step, 5 μL, of the supernatant,containing the ligation products, was transferred to a well of a PCRplate. Then 10 μL, of a PCR cocktail was added, containing 0.45 U Taqpolymerase, 0.6 μM P1 primer, 0.6 μM P2 primer, 1.5 mM MgCl₂, and 200 μMdNTPs. The thermocycler used the following program: 10 min at 94° C.,followed by 20 to 25 cycles of 30 sec at 94° C., 30 sec at 58° C., and30 sec at 72° C. The amplification products were then sequencedaccording to manufacturer's instructions. This representative ligationassay can be modified as in the following examples.

Example 2 Anchored Detector Designs

Upstream and downstream detector probe oligonucleotides were prepared asin FIGS. 2a and 3a for 24 target sequences identified as breast cancertargets: ACTB_1, TFF1_1, GATA3_3, GAPDH_3, CDH1_1, KRT19_2, TIMP1_2,NFKBIA_1, ESR1_1, VEGFA 3, LAMP1_2, MUC1_3, BAD_3, PTEN_1, BRCA2_1,BCAT2_3, ICAM1_2, IGF2_3, BRCA1_2, EGFR_1, BMP4_1, KIT_3, WNT1_1, andEGF_3 (in descending order of expected counts). The targets wereselected for a range of expression covering 6 orders of magnitude fromACTB_1 to EGF_3.

The assay was performed in triplicate with 100, 10, 1, and 0.1 and 0(control) nanograms of MCF7 total RNA as sample. The detectors wereadded to the sample in a volume of 1 or 2 and allowed to hybridize byincubating at 65° C. for 10 minutes, ramping down over 20 minutes from65° to 45° C., then held for 20 minutes at 45° C. Exonuclease I (E.coli) was added to the hybridization mixture in 6 μL of 0.5 Units andincubated for 1 hour at 37° C. T4 ligase was added to the mixture in 6μL of 5 Units and incubated for 1 hour at 37° C. A heat step wasperformed for 30 minutes at 80° C. The mixture was amplified by adding2× PCR master mix. The amplification products corresponding to thetarget sequences were detected and quantificated by qPCR and sequencing.

Example 3 Modified TempO-Seg™ Assay

Defining the nature of stochastic gene expression is important forunderstanding the regulation of transcription/translation and cellpopulation dynamics. Jurkat cells and human blood lymphocytes (activatedex vivo, fixed, permeabilized, antibody-stained for surface CD4 and CD8,and for intracellular transcription factors FoxP3 and EOMES) wereprepared. A modified version of whole transcriptome TempO-Seq™ geneexpression assay was performed in situ, and the cells were FACS-sortedinto bulk subpopulations or into single cells. In this modified version,the probes were eluted and gene expression was profiled by sequencing.The modified assay (based on the NIEHS S1500 gene-set) measured 2977genes (“surrogate whole transcriptome” or “surrogate” assay, compared tothe more comprehensive TempO-Seq “whole transcriptome” assay),identifying every known signaling pathway. Bulk cell measurementscorrelated with the summed single cell measurements (R²=0.89 for a bulkpreparation of 1000 CD4-/FoxP3-cells versus single cells). The no-samplecontrol background was <0.06 counts, showing that true “off” could bemeasured. The “abundance” of genes measured in bulk samples correlatedto the number of cells in which expression was “on”, a measure of thepercentage of time that the gene is on. Only 48 genes were expressed allthe time in every single cell, while the rest exhibited no expression inone or more cells. It was observed that most genes were either on or offwith very little “ramp up” or “ramp down” of expression over the timerequired to fix the cells and stop RNA synthesis/degradation.

If a simple average is used to compare the single-cell population to thebulk population, the expression behavior of individual cells over timemay be masked behind a single average value for the expression of thebulk population as a whole. When the bulk measurement was 10 counts, 247cells had 0 expression, 6 had a median expression of 500 (average 583),ranging from 149 to 1206 counts, compared to the highest expressed gene,average counts 12,541, range 7,519 to 18,970; only ˜16-fold higher.Thus, the concept of single copy gene expression is more complex thanpreviously understood. Rather, low-expressed genes are “off “most of thetime, but when “on” they are at relatively high levels in a cell. Thisin turn drives up “average” expression levels if measured in largerpopulations of nonactive cells.

FIG. 3 shows a modified version of the TempO-Seq™ assay that can beperformed after antibody-staining, before flow cytometry sorting (FACS).A reagent was used to permeabilize the cells, which provided highlysensitive antibody-staining of intracellular antigens. The protocol wascarried out by adding a cocktail of detector oligos (DOs) so that therewas a pair of DOs that hybridized to each targeted RNA, and whenproperly hybridized, the two detector oligos butt up against oneanother, permitting ligation. Wash steps were used to remove excessnonhybridized DOs, and subsequently, unligated DOs. The FACS sorting wasperformed, capturing each cell into 10 ml of PCR buffer, and thenuniversal PCR was carried out to amplify the products and at the sametime to add a sample-specific barcode to the product from each cell.

Example 4 Detection of Methylated Targets

A full-length mRNA for GAPDH has three target sequences GAPDH_1,GAPDH_2, and GAPDH_3, each target 50 bases in length. GAPDH_1 isupstream of a splice site, and has a position suspected of having an m¹Amodification at position 26, near a start codon. Pairs of detectors foreach of the three target sequences are provided, where performing theassay as disclosed herein can generate countable amplicons correspondingto GAPDH_1, _2, and _3 respectively, indicating those target sequencesare present in the mRNA sample. The count numbers may be adjustedquantitatively for minor count variations observed when detecting thethree targets, when the GAPDH targets are known to be present inequimolar amounts. However, the detectors for GAPDH_1 generate no (orsubstantially fewer) countable amplicons when the m¹A modification ispresent at position 26, compared to the expected counts with nomodification at position 26. The detectors for GAPDH_2 and _3 can thusserve as positive controls for the presence of the full-length mRNA,regardless of m¹A modification at position 26. Thus, the inventionprovides a method for detecting the presence of modifications, such asmethylation, at positions of interest in the nucleic acids of a sample.

Example 5 Processing FFPE Tissues Using the Standard TempO-Seq™ FFPEProtocol and Performance Profiling of H&E-Stained FFPEs

FFPE samples can be used in the standard TempO-Seq™ assay. In the FFPEpreparation protocol, the FFPE was unstained, antibody stained, or H&Estained. A 1-2 mm² area of a 5 μm thick section of FFPE was sufficient,making TMAs, core biopsies, FNAs suitable for assay. The sample can beslide mounted or a curl.

FFPEs from five prostate cancer patients were H&E stained. Then 1 mm²areas were identified for prostate: normal, adjacent high gradeprostatic intraepithelial neoplasia (PIN) or cancer epithelium. Theareas were scraped and processed through the standard TempO-Seq™ assayfor whole transcriptome. In FIG. 5, differential expression betweennormal and PIN versus normal and cancer was determined and plotted(log₂-fold change) for statistically significant genes (adjusted p-value<0.05). Most genes that were differentially expressed in cancer werealso differentially expressed in high grade PIN, indicating that at themolecular level, high grad PIN adjacent to cancer is in fact cancer insitu.

Example 6 Automated in Situ CellSensus™ Assay Process

The in situ TempO-Seq™ protocol was performed directly on slide-mountedFFPE tissue using an automated stainer (Bond RX, Leica BioSystems Inc.,Buffalo Grove, Ill.). As illustrated in FIG. 6, the FFPE sample wasdeparaffinized and processed by the automated stainer through the pointof detector oligo ligation. The automated stainer then stained theslides with antibodies (such as an anti-CD3 antibody) or optionally H&E(hematoxylin and eosin), performing (as desired) some of the stainingsteps manually, such as staining with eosine. The staining step includesimmunostaining. The CellSensus™ imaging platform was used to performpathological analysis and to image, and identify, select and/or markareas for profiling.

The imager then automatically recovered probes from those areas andtransferred them into PCR tubes that were processed through theremaining steps of the assay protocol described herein, includingamplification, qPCR, and sequencing. The data was analyzed byTempO-SeqR™ software to generate a report. Any number of imagingplatforms could have been used with appropriate hardware for elution,such as a capillary with fluidic control for applying the elution bufferto the surface of the sample.

Example 7 Single Cell Sensitivity

MCF-7 cells were processed through the in situ TempO-Seq™ WholeTranscriptome assay, then separated either by fluorescence-activatedcell sorting (FACS) or Cytospin™ cytocentrifuge (Thermo FisherScientific, Waltham, Mass.). The Cytospin-separated cells were thenpicked by the CellSensus™ system. In FIG. 7, panel (A) shows correlationof an assay of bulk 200 cells versus a single FACS-sorted cell. Panel(B) shows the correlation of the same 200-cell bulk and a single cellprofiled using the CellSensus™ instrument. Panel (C) shows correlationof one single cell isolated by FACS versus a single cell isolated by theCellSensus™ instrument. Stochastic gene expression was observed insingle cells, with genes measured as expressed in bulk but not expressedin some of the individual single cells. Panel C shows genes that wereexpressed by one single cell but not another, and vice versa.Low-expressed genes were nevertheless measurable from single cellsregardless of how they were picked, whether by FACS or by theCellSensus™ instrument.

Example 8 Focal Elution from FFPE Samples

Breast FFPE was processed through the in situ assay on the Bond RX, thenH&E stained. Areas of interest for profiling were digitally marked whileperforming IHC. The CellSensus™ instrument then carried out automatedelution. A reagent in the eluent destained the exposed area, providing apositive record of the area profiled. This is evident from thepre-elution and post elution images in FIG. 8. The intensity of the bluestaining was scanned in the pre- and post-elution images, clearlydemonstrating the destaining and the ability of the CellSensus™ imagerto assess and quantify the area from which the profiling data wasobtained.

The CellSensus™ assay of H&E-stained breast cancer epithelium wascompared to a 1 mm² area of scraped tissue (cancer and non-cancer), bothafter being processed on the Bond RX platform using the In Situ assaywith a targeted breast cancer panel of 486 genes. Table 1 below comparesthe counts for genes with greater than 5000 counts (1st column),demonstrating that the assays correlate for some genes, but that thenon-cancer tissue made a significant contribution, which the spatialresolution of the CellSensus™ assay addresses, reflected in the ratio(4th column) of CellSensus (2nd column) to scraped counts (3rd column).

TABLE 1 CellSensus Scraped Ratio MLPH 47728 129773 0.4 ESR1 20216 27407.4 TGFB3 13275 2417 5.5 RPLP0 12566 10820 1.2 MDM4 11102 11494 1.0UCHL5 10990 2781 4.0 PGR 10980 4797 2.3 YWHAB 10626 1323 8.0 SCUBE210131 1090 9.3 TRFC 10029 1716 5.8 CDH1 9404 7482 1.3 CDK4 8275 8623 1.0WNT5A 8247 3591 2.3 GRB7 7585 1207 6.3 VEGFA 7361 1192 6.2 ERBB2 44033007 1.5

Example 9 Differentially Expressed Genes Between Cell Lines andHistologic Transitions

A cell pellet mixture of MCF7 and Jurkat cells was fixed, embedded, andsectioned. Slides were processed through the in situ assay and thenstained with an anti-CD3 antibody and hematoxylin. This staining wasused to direct the selection of cells for gene expression profiling, forexample a cluster of CD3 negative cells. Table 2 provides counts for thehighest overexpressed genes in Jurkat (top set) and MCF7 (bottom set)for cell-type specific profiling directed by the antibody staining andIHC analysis.

TABLE 2 gene name MCF7 counts Jurkat counts Jurkat set: TSLP 0 170 GDF1552 154 SUPV3L1 2 183 BLMH 26 106 ASAH1 0 145 ICMT 1 300 RRS1 0 76 FGR 0316 PDHX 0 119 MCF7 set: ESR1 305 1 TFF1 392 2 SLC6A14 166 2 SPDEF 104 0PPIC 102 0

Profiling of 130 mm diameter areas of cancer and normal epithelium andstroma of prostate (Table 3) was carried out, as depicted FIG. 9, wherethe spatial resolution provided molecular specificity of biomarkers.

The ratio of detection between different cells, which can be spatiallyseparated by imaging or histologically distinguished, can be 1:10,1:100, 1:500, 1:1000, 1:2000, 1:5000 or greater. Where a marker isdetected in a cancer cell and there is no (or negligible) detection in anormal or stromal cell, or vice versa, the methods of the invention canbe said to provide absolute specificity.

TABLE 3 biomarker cancer normal stroma MALAT1* 768539 255266 110984DDX5* 10190 13909 5560 HNRNPA1* 8272 319 0 MT-ND6* 6209 8050 5363 EIF3E*4650 0 1256 MLPH* 4293 2 0 RPS7* 4037 0 0 ELK4 3982 3728 0 PTP4A1* 39530 0 MALT1* 3480 0 0 ABCC4^(†) 3317 0 0 CDH1 3253 3349 0 HPN* 3227 0 0SPDEF* 3135 0 0 RNF167* 3050 0 0 TSC22D1 2905 0 0 AKT2* 2885 0 0 CALR*2807 0 2 KLK2^(‡) 2793 0 0 CAMP* 2715 0 0 FAM213A* 2515 0 0 RNF4* 2463 00 EBNA1BP2* 2332 0 0 APH1A* 2238 0 0 IER2* 2216 0 0 SUZ12* 2179 0 0USO1* 2086 0 0 MAX* 2052 0 0 EPHB6* 2043 0 0 SAT1 0 3544 0 SOCS4 0 35060 NOP56 0 3130 0 Biomarkers with an asterisk (*) have previously beenassociated with prostate cancer. ABCC4 (†), also known as MRP4, is amultidrug resistance gene associated with androgen signaling that pumpsdrugs out of cells. KLK2 (‡) is the gene for Kallikrein 2, secreted bythe prostate in cancer (together with PSA produced by KLK3), and is animportant diagnostic marker.

The headings provided above are intended only to facilitate navigationwithin the document and should not be used to characterize the meaningof one portion of text compared to another. Skilled artisans willappreciate that additional embodiments are within the scope of theinvention. The invention is defined only by the following claims;limitations from the specification or its examples should not beimported into the claims.

We claim:
 1. A method for detecting a nucleic acid sequence from aselected area of a sample in situ, comprising in any order: imaging thesample for the presence or absence of an analyte; selecting an area ofthe sample less than 2 mm² based on the imaging; detecting a targetnucleic acid sequence having a downstream region (DR) and an upstreamregion (UR), by (1) contacting at least the selected area of the samplewith a downstream detector oligo (DDO) comprising a DR′ portion that iscomplementary to the DR, and an upstream detector oligo (UDO) comprisinga UR′ portion that is complementary to the UR, (2) ligating the DR′ andUR′ if both are specifically hybridized to the DR and UR of a targetsequence, and (3) eluting the ligation products from the selected areaby addition of a basic elution fluid, whereby the ligation productindicates the presence of the target sequence in the selected area. 2.The method of claim 1, wherein the area is selected is a morphologicalfeature.
 3. The method of claim 1, wherein the sample is from an FFPE.4. The method of claim 1, further comprising the step of deparaffinizingthe sample.
 5. The method of claim 1, further comprising the step ofanalyte retrieval.
 6. The method of claim 5, wherein the analyte is anantigen or nucleic acid.
 7. The method of claim 1, wherein the sample isimaged by histological stains.
 8. The method of claim 1, wherein thesample is imaged by immunostaining.
 9. The method of claim 1, whereinthe sample is imaged with a histological stain and by immunostaining.10. The method of claim 1, wherein the sample is dried after performinga step.
 11. The method of claim 1, wherein a wash step is performedafter detection steps (1) or (2).
 12. The method of claim 1, wherein theselected area is less than 0.2, 0.02, or 0.002 mm².
 13. The method ofclaim 1, wherein the target sequence is a portion of a cancer marker.14. The method of claim 1, further comprising the step of reimaging theselected area, to verify collection of ligation products.
 15. A methodfor detecting a neoplastic state of a cell by performing the method ofclaim 1 where a first cancer marker sequence is detected in the cell.16. The method of claim 15, wherein the number of ligation productsdetected for a second cancer marker sequence is less than 10%, 1%,0.01%, or 0.05% of the number of ligation products detected for thefirst cancer marker sequence.
 17. The method of claim 16, wherein acancer marker is detected a neoplastic cell and not detected in a normalcell, providing absolute specificity.
 18. A method for generating a geneexpression profile for a selected area, comprising performing the methodof claim 1 for a plurality of target sequences.
 19. A method fordetecting a neoplastic state of a cells in a tissue by performing themethod of claim 18 for a plurality cancer marker sequences on cells intwo separate areas of the tissue.
 20. A method for diagnosing a diseasestate by performing the method of claim 1, wherein the target sequenceis detected in the area of a morphological feature.
 21. A kit comprisingthe detector oligos of claim 1, a basic elution fluid, and a stain. 22.The method of claim 1, wherein elution step (3) comprises applying thebasic elution fluid to the selected area of the sample in situ.
 23. Themethod of claim 1, wherein elution step (3) comprises applying the basicelution fluid to the selected area of the sample using a capillary withfluidic control.
 24. The method of claim 1, wherein elution step (3)comprises recovering the ligation products from the selected area of thesample in situ.
 25. The method of claim 1, wherein elution step (3)comprises transferring the eluted ligation products using a capillarywith fluidic control.
 26. The method of claim 1, wherein elution step(3) comprises transferring the eluted ligation products to a container.